A quantitative real time PCR method to analyze T cell receptor Vbeta subgroup expansion by staphylococcal superantigens.

BACKGROUND

Staphylococcal enterotoxins (SEs), SE-like (SEl) toxins, and toxic shock syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus, belong to the subgroup of microbial superantigens (SAgs). SAgs induce clonal proliferation of T cells bearing specific variable regions of the T cell receptor beta chain (Vbeta). Quantitative real time PCR (qRT-PCR) has become widely accepted for rapid and reproducible mRNA quantification. Although the quantification of Vbeta subgroups using qRT-PCR has been reported, qRT-PCR using both primers annealing to selected Vbeta nucleotide sequences and SYBR Green I reporter has not been applied to assess Vbeta-dependent expansion of T cells by SAgs.

METHODS

Human peripheral blood mononuclear cells were stimulated with various SAgs or a monoclonal antibody specific to human CD3. Highly specific expansion of Vbeta subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers corresponding to selected Vbeta nucleotide sequences.

RESULTS

qRT-PCR specificities were confirmed by sequencing amplified PCR products and melting curve analysis. To assess qRT-PCR efficiencies, standard curves were generated for each primer set. The average slope and R2 of standard curves were -3.3764 +/- 0.0245 and 0.99856 +/- 0.000478, respectively, demonstrating that the qRT-PCR established in this study is highly efficient. With some exceptions, SAg Vbeta specificities observed in this study were similar to those reported in previous studies.

CONCLUSIONS

The qRT-PCR method established in this study produced an accurate and reproducible assessment of Vbeta-dependent expansion of human T cells by staphylococcal SAgs. This method could be a useful tool in the characterization T cell proliferation by newly discovered SAg and in the investigation of biological effects of SAgs linked to pathogenesis.